Alkaline Phosphatase Encapsulated in Gellan-Chitosan Hybrid Capsules

Alkaline Phosphatase Encapsulated in Gellan-Chitosan

Hybrid Capsules

Toshihiro Fujii,*1 Daisuke Ogiwara,1 Kousaku Ohkawa,2 Hiroyuki Yamamoto2

1 Department of Kansei Engineering, Faculty of Textile Science and Technology, Shinshu University, Tokida 3-15-1,Ueda 386-8567, JapanFax: (þ81) 268-21-5518; E-mail: [email protected]

2 Institute of High Polymer Research, Faculty of Textile Science and Technology, Shinshu University, Tokida 3-15-1,Ueda 386-8567, Japan

Received: November 10, 2004; Revised: February 9, 2005; Accepted: February 10, 2005; DOI: 10.1002/mabi.200400190

Keywords: biological applications of polymers; encapsulation; enzymes; gellan-chitosan; polyion complex capsule

Introduction

An encapsulation system containing biological components,

such as enzymes, proteins and detoxicants, was originally

proposed by Chang as an ‘‘artificial cell’’.[1] The concept of

the artificial cell has now been developed into diverse

biological encapsulated formulations, for example, micro-

encapsulated invertase,[2] glucose oxidase,[3] cytochrome

C,[4] fungal proteases,[5] asparaginase and catalase.[6] The

benefits of encapsulating and releasing a therapeutic agent

from a polymer matrix include protecting the sustained

release of the encapsulated substances.[7] The use of micro-

capsules and microspheres is very promising because of

their small particle size, which enables repetitive adminis-

tration as a therapeutic bolus by either an injection or an oral

route. Biocompatible and biodegradable materials, in-

cluding natural polysaccharides such as chitin, chitosan,[8]

alginate,[9] xanthane[10] and synthetic biodegradable poly-

mers such as poly(lactic-co-glycolic acid) and poly(lactic-

co-amide), have been thoroughly investigated for this

purpose.

The selection of the methodology for encapsulation is

rather important in order to achieve biological encapsula-

tion in which the enzymes or peptides retain their catalytic

activity or biological function fully. A preparative method

by complex coacervation is based on polyionic complexa-

Summary: Alkaline phosphatase (ALP) was encapsulatedin gellan-chitosan polyion complex (PIC) capsules using aconvenient procedure. The recovery of ALP was about 50%when the capsules were prepared by dropping a solutionof ALP and gellan mixture (ALP/gellan) into a chitosansolution. When p-nitrophenyl phosphate (p-NPP) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) were incubatedwith ALP/gellan-chitosan capsules as substrates for ALP, thetransparent colorless capsules changed to yellow and blue,respectively. The encapsulation of ALP into the PIC capsuleswas also confirmed by SDS-PAGE and immunoblot analyses.The ALP and polypeptides of more than 30 kDa remainedwithout release even after incubation at 4 8C for 14 d. Thebiochemical properties of the encapsulated ALP activitywere similar to those of the intact enzyme. When the solutioncontaining p-NPP was loaded on a column packed with ALP/gellan-chitosan capsules at 27 8C, approximately 75% ofp-NPP was hydrolyzed by passing through the column. Nosignificant leakage of ALP was observed during the proce-dure, indicating that the capsules were resistant to pressurein the chromatographic operation. Furthermore, 70% of thehydrolytic activity of the packed capsules remained afterstorage at 4 8C for one month. These results suggest that

the polyion complex capsules could be useful materials forprotein fixation without chemical modification.

Encapsulation of ALP into PIC capsules and the morpholo-gical changes seen in the absence of the ALP substrate and inthe presence of p-NPP and BICP.

Macromol. Biosci. 2005, 5, 394–400 DOI: 10.1002/mabi.200400190 � 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

394 Full Paper

tion[11,12] through electrostatic interactions between catio-

nic and anionic polymers, resulting in the formulation of

insoluble spherical capsules. Recently, we have reported the

preparation and characterization of several kinds of poly-

ion complex (PIC) capsules, including gellan-chitosan,[13]

poly(L-lysine)-gellan,[14] chitosan-poly(L-glutamic acid)[15]

and poly(L-lysine)-poly(L-glutamic acid).[16] More re-

cently, the membrane permeability and drug releasing

properties of the gellan-chitosan capsule have been reported

in detail.[17] The gellan-chitosan capsule retains proteins

inside, while releasing low-molecular-weight substances

across the capsule membrane. For instance, two proteases,

trypsin and a-chymotrypsin, have been successfully encap-

sulated into gellan-chitosan capsules. When bovine serum

albumin (BSA) was encapsulated with one of these pro-

teases, the hydrolyzed fragments of BSA were gradually

released from the gellan-chitosan capsule, while undigested

BSAwas retained in the capsule. These results indicate that

the gellan-chitosan capsule is very promising as a carrier

material for active enzymes.[18]

As the second test of the enzyme encapsulation, we

chose alkaline phosphatase (ALP) as the encapsulant in

the gellan-chitosan capsule. ALP is a ubiquitous, stable

enzyme which has been widely applied as a ‘‘proving’’

material in biochemistry, immunological chemistry, mor-

phology and cell biology. The catalytic activity of ALP

can be easily measured and several groups have report-

ed the ALP-sol-gel encapsulating systems as biosensing

devices.[19,20] In the present study, we discuss (i) the

encapsulation of ALP into gellan-chitosan PIC capsules;

(ii) the biochemical characterization of the encapsulated

ALP and (iii) the application of ALP-encapsulating PIC

capsules as a bio-preparative reactor.

Experimental Part

Materials

Chitosan-10, -100, -500, and -1000, gellan, p-nitrophenylphosphate (p-NPP) and horseradish peroxidase-conjugatedgoat anti-mouse antibodies were purchased from Wako PureChemical Industry. The average molecular weights ofchitosan-10, -100, -500 and -1000 were 2.1� 105, 13.1�105, 15.8� 105 and 18.0� 105, respectively.[13] Purified andcrude ALP from porcine intestine and anti-alkaline phos-phatase monoclonal antibodies were purchased from Sigma. 5-Bromo-4-chloro-3-indolyl phosphate (BCIP) was obtainedfrom KPL. A low-molecular-weight protein standard wasobtained from Pharmacia. All other chemicals used were ofreagent grade.

Encapsulation of ALP into Gellan-Chitosan Capsules

The gellan-chitosan capsules were prepared as described ina previous publication.[17] Briefly, 1% (weight per volumepercentage; w/v) gellan solution (in H2O) containing 0–2mg �mL�1 ALP was dropped into 0.5% (w/v) chitosan solution

(in 150� 10�3M acetic acid, pH 2.5–3.0). After standing for

5–10 min at room temperature, the formed capsules werewashed with TBS (Tris-buffered saline: 20� 10�3

M Tris-HCl,137� 10�3

M NaCl, pH 7.5) three times and stored at 4 8C priorto use.

Assay of ALPActivity

In the standard assay, the ALP-encapsulating capsules in1.8 mL of a buffer solution (40� 10�3

M Tris-HCl, 1� 10�3M

MgCl2, pH 8.5) were pre-incubated at 37 8C for 5 min. Thereaction was then initiated by adding 200 mL of the substratesolution (100� 10�3

M p-NPP). The ALP began to hydrolyzep-NPP to p-nitrophenol (p-NP). After incubation at 37 8C for0–60 min, 300mL of the liquid was pipetted out and transferredinto a test tube. The enzyme reaction was then terminated bythe addition of 100 mL of 3 M NaOH. After flash centrifugation,the absorbance of the supernatant was measured at 405 nm.One unit of ALP was defined to be the amount needed tohydrolyze 1 mmole of p-NPP per min at 37 8C.

Electrophoresis and Immunoblot Analysis

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) was conducted according to the method ofLaemmli[21] with a 13.5% slab gel. Tricine/SDS gel electro-phoresis was carried out on 10/20% discontinuous polyacryl-amide gel, as described by Schagger.[22] Proteins in the gelwere stained with 0.1% Coomassie brilliant blue R-250, 10%acetic acid and 40% ethanol for 1 h and destained in 10% aceticacid and 40% ethanol. For immunoblotting, proteins wereelectrophoretically transferred from the SDS-polyacrylamidegel onto a nitrocellulose membrane. The membrane wasblocked with a 1% bovine serum albumin solution in10� 10�3 M Tris-HCl, pH 7.4, containing 150� 10�3

M NaCland incubated with a 1:1 000-fold dilution of anti-ALP mono-clonal antibodies, then treated with a 1:3 000-fold dilutionof horseradish peroxidase-conjugated goat anti-mouse anti-bodies. After immunoreaction, the membrane was washed witha buffer solution (10� 10�3

M Tris-HCl, 150� 10�3M NaCl,

0.5% Tween-20, pH 7.4). The immunoreactive bands weredeveloped using 4-chloro-1-naphthylphosphate and H2O2 asthe substrate for the horseradish peroxidase.[23,24]

Results and Discussion

Encapsulation of ALP intoGellan-Chitosan Capsules

The encapsulation procedure is illustrated in Figure 1.

The ALP was mixed with the dropping solution and added

dropwise to the receiving solution. Immediately, the poly-

ionic complexation reaction between chitosan and gellan

occurred at the interface, thus producing true spherical

capsules which retained ALP inside. The encapsulated ALP

activity was defined as the recovered percentage of the ALP

activity in the capsules towards the total ALP activity in the

dropping solution.

Alkaline Phosphatase Encapsulated in Gellan-Chitosan Hybrid Capsules 395

Macromol. Biosci. 2005, 5, 394–400 www.mbs-journal.de � 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

First, we examined the recovery of the ALP activity after

different encapsulating procedures. The molecular weight

of chitosan is thought to be important for the formation of

the PIC capsules.[17] We prepared gellan-chitosan PIC

capsules incorporating ALP using two kinds of experi-

mental combinations, by exchanging cationic chitosan and

anionic gellan as the dropping and receiving solutions.

When the ALP-gellan mixed (ALP/gellan) solution was

added dropwise to the chitosan-10 and -100 solutions,

transparent spherical capsules with a diameter of 3–5 mm

were immediately formed, as reported previously.[13,17]

After standing for 20–30 min and washing with TBS,

the ALP activity of the capsules was measured (Table 1).

The recovery of ALP activity was 53.8� 6.2% and

49.4� 10.1% for chitosan-10 and -100 as the receiving

solutions, respectively. Approximately 30–40 capsules

were prepared from 1 mL of dropping solution. The results

were consistent when gellan or chitosan were used as the

receiving solution. The opposite addition order of chitosan-

10 or -100 (dropping) and gellan (receiving) was not

effective for the formation of capsules under the conditions

used, because of their different densities. The pH values

of the chitosan-10 and -100 solutions are in the pH range

2.5–3.0, and when the chitosan solutions were mixed with

ALP and used as the dropping solutions, the recovery

percentages of the ALP activity were 34.2� 7.1% and

35.1� 6.5% for chitosan-10 and -100, respectively. These

values were lower than those in the cases when gellan

solution (pH 6.8) was used as the dropping solution. This

may be due to the exposure of the enzyme to a low pH

solution (150� 10�3M acetic acid), resulting in the partial

denaturation of ALP. Thus, the lower pH values of the

chitosan solution possibly limit the encapsulation of several

kinds of acid-labile enzymes, and in such cases, use of

gellan solution as the dropping solution would improve the

recovery percentages of encapsulated enzyme activities.

Characterization of Encapsulated ALP

The recovery of ALP activity in the gellan-chitosan PIC

capsules as a function of ALP concentration is shown in

Figure 2(a). For this experiment, crude and purified ALP

were employed. When crude ALP (0.1–3.0 units) was

mixed with a 1% gellan solution (1 mL) and then dropped

into the chitosan-10 solution, the recovery of the encapsu-

lated ALP activity was 50–60%. On the other hand, the

value was lower (15–25%) when purified ALP (0.1–

3.0 units) was used. Both in the case of crude and purified

ALP, the recovery percentages were highest at 0.6 units and

then decreased with increasing ALP concentration. The

spherical capsules obtained at all the ALP concentrations

tested were stable to mechanical stimuli (magnetic stirring).

Hence in the following experiments, we used a combination

of 1% gellan/ALP (0.6 units per mL of gellan solution) and

0.5% chitosan-10.

Addition of p-NPP and BCIP, substrates of ALP, pro-

duced bright yellow colored and blue colored capsules,

respectively (Figure 2(b)), indicating that the encapsu-

lated ALP remained active. Time courses of p-NPP

hydrolysis by intact and encapsulated ALP are represented

in Figure 2(c). An initial lag in p-NP release was found

with the encapsulated ALP. This delay will be spent in

the diffusion of p-NPP (molecular weight¼ 263) and p-NP

(molecular weight¼ 139) across the membrane of the

gellan-chitosan PIC capsules. After incubation for 5 min at

37 8C, the hydrolysis of p-NPP proceeded at a constant rate

for at least 60 min. The Michaelis-Menten constant, KM

value, was measured for each of the intact and encapsulated

enzymes. When p-NPP was used as the substrate,KM values

Figure 1. Encapsulation of alkaline phosphatase (ALP) intogellan-chitosan polyion complex (PIC) capsules.

Table 1. Recovery of alkaline phosphatase activity duringencapsulation.

Dropping solution(containing ALP)a)

Receivingsolutiona)

Recovery ofALP activityb)

%

Gellan Chitosan-10 53.8� 6.2Gellan Chitosan-100 49.4� 10.1Gellan Chitosan-500 n.f.c)

Gellan Chitosan-1000 n.f.Chitosan-10 Gellan n.f.Chitosan-100 Gellan n.f.Chitosan-500 Gellan 34.2� 7.1Chitosan-1000 Gellan 35.1� 6.5

a) Defined in Figure 1.b) Encapsulated alkaline phosphatase activity value calculated

from the total activity in the dropping solution (100%).c) Capsules were not formed.

396 T. Fujii, D. Ogiwara, K. Ohkawa, H. Yamamoto


were 0.1–0.11� 10�3M and 0.14–0.18� 10�3

M for the

intact and encapsulated ALPs, respectively. The KM value

of the encapsulated ALP was slightly higher than that of

the intact ALP, suggesting that the active center of the

encapsulated ALP was in a similar state to that of the intact

enzyme.

The biochemical properties of the encapsulated ALP

activity were compared with those of the intact enzyme

(Table 2). The intact and encapsulated ALP activities

showed an absolute requirement for divalent cations such as

Mg2þ and Ca2þ, while chelation of the divalent cations by

EDTA significantly suppressed the ALP activities. Both

intact and encapsulated ALP activities were observed with

similar sensitivities over wide pH and temperature ranges.

These data suggest that ALP behaves like a free enzyme

after encapsulation into the capsules.

Identification of EncapsulatedALP by Immunoblotting

The encapsulated proteins in the PIC capsules were re-

extracted with sample buffer (31� 10�3M Tris-HCl, 1%

SDS, 5% glycerol, 0.15% 2-mercaptoethanol, 0.0005%

pyronin Y, pH 6.8) and the protein compositions were

analyzed by SDS-PAGE (Figure 3(a)). For the crude

ALP preparation, several protein bands with molecular

masses of 14–100 kDa were stained with Coomassie-blue

(Figure 3(a), lane 1, 2). Purified ALP showed a single

polypeptide with a molecular mass of 67 kDa (Figure 3(a),

lane 3, 4). Immunoblot analysis using anti-ALP monoclonal

antibodies confirmed that the 67 kDa bands in both the

crude and purified ALP preparations were actually porcine

Figure 2. Characterization of ALP-encapsulating gellan-chito-san PIC capsules. (a) Relationship between the ALP concentrationand the recovery of ALP activity in the PIC capsules: * crudeALP;*purifiedALP. (b)Morphological observationofALP-encapsulating gellan-chitosan PIC capsules in the absence(left) of the ALP substrate and in the presence of p-NPP(middle) and of BICP (right). (c) Time course of p-NP liberationfrom the ALP-encapsulating PIC capsules: * intact ALP; *ALP/gellan-chitosan capsules.

Table 2. Effects of various factors on alkaline phosphataseactivities in intact and encapsulated states.

Factors Intact Encapsulateda)

None (control)b) 100.0 100.010� 10�3

M EDTA 3.7� 1.8 3.7� 1.83� 10�3

M MgCl2 104.1� 4.3 114.2� 14.6CaCl2 98.1� 7.5 96.5� 6.4pH 8.0 65.3� 4.8 74.5� 5.8pH 9.0 169.0� 8.5 144.2� 10.8pH 10.0 171.6� 4.0 153.7� 10.5Temperature: 25 8C 74.0� 1.7 78.5� 7.6Temperature: 35 8C 97.2� 3.5 96.0� 6.5Temperature: 45 8C 150.4� 5.6 141.7� 9.5Temperature: 55 8C 118.5� 9.7 141.7� 5.1

a) Encapsulated in gellan-chitosan PIC capsule.b) Measured using the standard assay conditions, as described in

the Experimental Part.

Figure 3. Identification of ALP in gellan-chitosan PIC capsules.PIC capsules were incubated with an equal volume of samplebuffer at 95 8C for 2 min. After centrifugation at 1 000� g for3 min, the obtained supernatants were subjected to 13.5% SDS-PAGE. The separated proteins were transferred onto a nitrocel-lulose membrane: (a) Coomassie brilliant blue staining; (b)immunostaining with anti-ALP monoclonal antibody. Lane 1:crude ALP; Lane 2: crude ALP in the gellan-chitosan capsules;Lane 3: purified ALP; Lane 4: purified ALP in the gellan-chitosancapsules; Lane 5: ALP-free gellan-chitosan capsules.



intestine ALP (Figure 3(b), lane 1–4). The pre-encapsu-

lated protein (Figure 3(b), lane 1) and the re-extracted

protein (post-encapsulated; Figure 3(b), lane 2) exhibited

identical patterns in their immunoreactive bands. The

immunoreactive bands of the crude preparation were found

mainly at 67 kDa and 95 kDa, as reported previously

(Figure 3(b)).[24] The 67 kDa polypeptide of the crude ALP

strongly cross-reacted with the antibodies. These results

indicate that ALP was successfully encapsulated into the

gellan-chitosan PIC capsules in the stable state. Further-

more, proteins with low molecular weights were also

trapped in the PIC capsules. The release of the proteins from

the PIC capsule was examined in the following experiment.

Permeability of Gellan-Chitosan Capsules

The permeation of the encapsulated inner materials will be

mainly dependent on their molecular masses, molecular

structures and electrical charges. In order to examine the

molecular weight dependence of the protein release from

the PIC capsules, the following proteins were encapsulated

into gellan-chitosan capsules: phosphorylase b (94 kDa);

bovine serum albumin (67 kDa); ovalbumin (43 kDa);

carbonic anhydrase (30 kDa); soybean trypsin inhibitor

(20.1 kDa); a-lactalbumin (14.4 kDa). After incuba-

tion for 0–10 d at 4 8C in TBS, the remaining proteins

in the capsules were analyzed by Tricine/SDS-PAGE

(Figure 4(a)). The proteins with molecular masses of more

than 30 kDa remained in the capsules even after 10 d. On the

other hand, a-lactalbumin (14.4 kDa) and soybean trypsin

inhibitor (20.1 kDa) were gradually released from the

capsules, and the remaining proteins were decreased to 50%

after 5–6 h for a-lactalbumin and after 1 d for soybean

trypsin inhibitor. Similar results were observed over a wide

pH range (pH 5–9) and at a high salt concentration

(1 M NaCl) in TBS (data not shown). These results suggest-

ed that the exclusion limit of the gellan-chitosan capsules

used was about 30 kDa. This value is almost in accord with

that obtained with the gellan-chitosan capsules we reported

on previously.[17]

Application of ALP/Gellan-ChitosanCapsules as a Bioreactor

We prepared a column (f 1� 16.5 cm) packed with ALP/

gellan-chitosan capsules, each of which contained 50 units

of ALP. The experiments were performed 1 d and 30 d after

capsule preparation. The column was equilibrated with

a buffer solution (40� 10�3M Tris-HCl, 1� 10�3

M MgCl2,

pH 8.5) at 27 8C, then 80 mL of buffer containing 2� 10�3M

p-NPP was applied to the column at a flow rate of

18 mL � h�1. As shown in the results in Figure 2(b), the

color of the column changed from transparent to light

yellow (Figure 5(a)). The yellow color lasted for 2.5 h until

the p-NPP containing buffer was replaced by the substrate-

free buffer. This elution profile is represented in Figure 5(b).

More than 70% of the total p-NPP applied was hydrolyzed

in the elution volume from 15 to 60 mL. No protein was

detected in the collected fractions by means of SDS-PAGE

and immunoblot analyses (data not shown), indicating that

the proteins did not leak out of the capsules during the

chromatographic procedure. After storage of the column

at 4 8C for 30 d, the same experiment was carried out.

Approximately 50% of the total p-NPP applied was hydro-

lyzed, and the hydrolyzing activity corresponded to 70%

of the original level (after 1 d) of the column. These results

indicate that gellan-chitosan PIC capsules encapsulating

ALP and other enzymes will function as a bioreactor.

The ALP capsules were dried out and stored at room

temperature. Then the dried ALP capsules were swollen

again in TBS to reconstitute the pre-dried state, and the

reconstituted ALP capsules recovered their catalytic

activity to at least 80% (data not shown). This result

indicates the possibility of repeated use of the ALP in the

gellan-chitosan capsules.

Figure 4. Relationship between the molecular weights of theencapsulated proteins and the release kinetics from gellan-chitosan capsules: (a) SDS-PAGE of the remaining protein in thegellan-chitosan PIC capsule; (b) Time course of the remainingprotein (%), which was determined by densitometry of the proteinbands: * phosphorylase b (94 kDa);* bovine serum albumin(67 kDa); ~ ovalbumin (43 kDa); ~ carbonic anhydrase(30 kDa); ^ soybean trypsin inhibitor (20.1 kDa); } a-lactalbumin (14.4 kDa).



Conclusion

Since early investigations for creating polymeric encapsu-

lating materials, three major approaches have been con-

firmed: (i) complex coacervation; (ii) phase separation;

(iii) in situ polymerization. The present method adopted by

this group, utilizing the polyionic complexation reaction

between the counter-charged polysaccharides, is classified

as (iii). More recently, sol-gel methods[25] have been devel-

oped as a versatile encapsulating methodology for biolo-

gical compounds (reviewed in ref.[20]). For the purpose of

encapsulating an enzyme and fully retaining its catalytic

activity in the capsule, the encapsulation should essentially

be performed in aqueous solution. In this sense, both our

present method and the sol-gel method are applicable for

biological encapsulants. Usually, the sol-gel precursors are

SiO2 and the encapsulants are immobilized in a porous gel

network around the encapsulant molecules. The sol-gel

method receives much attention from researchers whose

interests are directed towards biosensing devices or re-

cycleable enzymes for a specific organic synthesis.[20]

On the other hand, the polyionic complexation method is

appropriate for the medical, agricultural and biochemical

industry because, as we have demonstrated in the present

study, chitosan and gellan are both biodegradable and bio-

compatible materials. For example, enzyme-encapsulating

gellan-chitosan capsules can be implanted directly into the

bodies of organisms, eliminating the need for surgical re-

moval after use of the material due to their bio-resorbability.

Thus the sol-gel method and the polyionic complexation

methods have different advantages for the applications of

the encapsulating formulations.

The isolation of biopolymers, tissues and cells from the

host body system is necessary to protect the implanted

polymers from protease and immune systems. Encapsu-

lation is thought to be an effective technique for solving

this problem. Both gellan and chitosan are natural

biopolymers and are widely utilized in food and medical

science, because of their safety and low price. The enzyme

encapsulating procedure described in the present study does

not need any chemical modifications and can be conveni-

ently performed in aqueous solution. The encapsulated

enzyme as described in this work fully retains its biological

catalytic activity. In conclusion, the chitosan-gellan PIC

encapsulating system will further inspire a broad spectrum

of applications and be a powerful tool in the field of

biotechnology.

Acknowledgements: We wish to thank to Mike Honywood fora critical reading of the manuscript. This study was supportedby Grants-in-aid for the 21st Century COE Program and ScienceResearch (No. 14750709 for KO, No. 16350123 for TF,No.16651064 for HY) from The Ministry of Education, Culture,Sports, Science and Technology of Japan.

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Figure 5. Preparation of the bioreactor column packed withALP/gellan-chitosan capsules: (a) before (left) and after (right)loading the substrate (p-NPP) solution; (b) elution profile of p-NP,the hydrolysis product of p-NPP. The chromatography was carriedout using the same column stored for: * 1 d; * 30 d. Fractions(1 mL) were collected and the absorbances (405 nm) weremeasured.



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Alkaline Phosphatase Encapsulated in Gellan-Chitosan Hybrid Capsules